Hybridization of HI-labeled ribosomal RNA to human chromosomes on slides resulted in specific labeling of the satellite regions of chromosomes 13, 14, 15, 21, and 22, with an over-all efficiency of about 5%. Differences between D and G chromosomes, and between associated and unassociated satellites, were not significant. Labeling of all other parts of the preparations was nonspecific, and increased in the order: extrachromosomal regions < chromosome arms < centric regions.Three lines of evidence bear on the localization of rDNA in the human genome. First, the satellited chromosomes tend to be associated with the nucleolus (1, 2). Second, when isolated chromosomes are fractionated, enrichment for small chromosomes enriches for rDNA (3). Third, with DNA from cell lines having different ratios of acrocentric to total chromosomes, rRNA-DNA hybridization increases with the relative number of acrocentrics (4). Such results are consistent with the presence of rDNA in some combination of chromosomes 13, 14, 15, 21, and 22, but do not resolve whether all of these chromosomes, are uniquely involved.We have used the technique of in situ hybridization (5)

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Hepatitis B virus (HBV), a virus with known carcinogenic potential, integrates into cellular DNA during long-term persistent infection in man. Hepatocellular carcinomas isolated from viral carriers often contain clonally propagated viral DNA integrations. As small chromosomal deletions are associated with several types of carcinomas, the occurrence of chromosomal deletions in association with HBV integration in hepatocellular carcinoma was studied. HBV integration was accompanied by a deletion of at least 13.5 kilobases of cellular sequences in a human hepatocellular carcinoma. The viral DNA integration and deletion of cellular sequences occurred on the short arm of chromosome 11 at location 11p13-11p14. The cellular sequences that were deleted at the site of HBV integration were lost from the tumor cells, leaving only a single copy of the remaining cellular allele.

(11)(12)(13)(14)(15)(16)(17)(18). The episomal DNA is formed by covalent joining of the direct repeats that are at both ends of the 175,000-base-pair linear virion EBV DNA (14).Although episomal EBV DNA has been clearly demonstrated in latently infected cells, integrated EBV could also be present. The persistence of DNA in latently infected cells over years of active cell replication suggests the possibility of integration (15). Integration of EBV DNA not only could be important as a mechanism for persistence but also could result in alteration of virus or cell gene expression. A chromosome 8 to chromosome 2, 14, or 22 translocation is characteristic of malignant B lymphocytes, including those of African Burkitt lymphoma (16). One direct way in which EBV could promote Burkitt lymphoma is by integration at the site of the translocation.Integration of EBV DNA has been suggested by the detection of viral DNA at a density intermediate between EBV DNA and cell DNA (17). However, covalent linkage of cell and viral DNA has not been established (18). Attempts to identify an association between EBV DNA and specific human chromosomes by somatic cell hybridization have yielded inconclusive and conflicting results, possibly due to the persistence of episomal copies of EBV DNA in the cell hybrids (19)(20)(21)(22).We report here the cytological hybridization of recombinant EBV DNA probes to metaphase chromosomes from two latently infected human cell lines, Namalwa and IB4. Namalwa is a culture of lymphocytes from an African Burkitt tumor biopsy sample (7, 23). IB4 is a line of latently infected neonatal lymphocytes established by growth transformation in vitro of cord blood lymphocytes with EBV (17). Viral DNA localizes to unique and nonidentical chromosomal sites in the two cell lines. These sites are among the chromosome sites that have DNA homology to the IR3 simple repeat array in EBV DNA (24,25). Because virus-cell DNA homology could mediate integrative recombination at various chromosomal sites, we have investigated whether EBV IR3 recombined with homologous sequences of Namalwa or IB4 cells. MATERIALS AND METHODSLymphocytes, Chromosomes, and Cytological Hybridization. IB4, IB4 clone D, and Namalwa cells were grown in RPMI 1640 medium supplemented with 10% newborn calf serum (8). The IB4 cell line was established by infecting cord blood lymphocytes with a 10-4 dilution of B95-8 culture supernatant containing EBV (8). At the 10-4 and 10-3 dilutions, only one of four infected cultures yielded continuous cell lines in the IB experiment, suggesting that the IB4 cell line was likely to result from a single infected cell. IB4 cells were maintained in culture for 1 year before cloning in soft agarose to yield IB4 clone D (8,10). T cells for control hybridizations were prepared from the peripheral blood of human donors and stimulated with phytohemagglutinin (PHA) (25). Chromosomes were prepared from cells that were incubated in medium containing Colcemid (26). Chromosomes were Giemsa banded and photographed so that they could...